Human ovarian mesothelial cells and methods of isolation and uses thereof

ABSTRACT

The invention discloses a substantially pure population of human ovarian mesothelial cells and methods of isolating and culturing the ovarian mesothelial cells. By carefully manipulating the microenvironment of the ovarian mesothelial cells, multiple passages are attainable wherein the ovarian mesothelial cells are capable of becoming ovary surface epithelial cells or granulosa cells. In addition, several methods of use of human ovarian mesothelial cells are disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/545,659, filed on Apr. 10, 2000, which is incorporated herein byreference in its entirety.

TECHNICAL FIELD

This invention is in the field of developmental biology and cellbiology. Specifically, this invention relates to a population of ovarianmesothelial cells that are capable of differentiating into ovariansurface epithelial cells and granulosa cells, methods of isolating theovarian mesothelial cells, characterization of ovarian mesothelialcells, and uses of the ovarian mesothelial cells.

BACKGROUND ART

Ovarian cancer is one of the most common causes of death due to cancerin women. Boring C. C. et. al. CA-Cancer J. Clin. 41,19-36, (1991). Inabout 80-90% of ovarian cancer cases, the ovarian surface epithelium isthought to be the tissue source in which the cancerous transformationoccurs. Nicosia S. V. Pathology of Human Neoplasms. New York: RavenPress 435-486, (1988); Scully R. E. Am. J. Pathol. 87:686-720, (1977).The ovarian surface epithelium (OSE) is a single cell layer covering thesurface of the ovary and is contiguous with coelomic mesothelium at theovarian hilum. Dubeau L. et. al. Anticancer Research 10: 1233-1240,(1990). The OSE cells are thought to be derived from mesothelial cellscovering the gonadal ridge. Mesothelial cells stromalize, migrate insidethe ovary, and become granulosa cells. Granulosa cells, one of the majorendocrine cell types in the ovary, first appear in a primordial follicleas a single layer of cells surrounding an oocyte during prenatal life.As each follicle becomes active, the oocyte begins to enlarge in sizeand granulosa cells begin to divide, increase in number, and secreteestrogen. As the follicle matures, granulosa cells stop dividing and thefollicle is erupted to the oocyte in a process known as ovulation. Afterovulation, the granulosa cells then differentiate into luteal cells ofthe developing corpus luteum from which progesterone is secreted.

The lack of human ovary tissue models makes efforts to confirm theovarian surface epithelium as the source of cancerous growth difficult.Further, there is a lack of understanding about the development ofovarian surface epithelial cell during oogenesis and follicle formationbecause culturing conditions in which the OSE cells can be isolated andstill retain their original characteristics are still being refined. Theidentification, isolation, and characterization of an ovarian precursorcell that can reliably differentiate into multiple types of cells, suchas OSE cells and granulosa cells, may be important in the understandingof the biology of ovarian cells and why cancerous transformations occurlike in ovarian cancer and perhaps how the transformations can beprevented. However, the ovarian precursor cells, like many precursorcells, are few in number and difficult to isolate. Once isolated,ovarian precursor cells are difficult to culture to the extent that theyretain their precursor traits. It is postulated that there areprecursors to granulosa cells and OSE cells, however, the workaccomplished to date only isolates OSE cells and granulosa cells but nota common precursor to both types of cells.

There have been several reports of isolation and culturing methods ofhuman OSE cells. Kruk P. A. et. al. Laboratory Investigation 63(1),132-136, (1990); Siemens C. H. and Auersperg N. Journal of CellularPhysiology 134, 347-356, (1988); Auersperg N. et. al. In Vitro 20(10)743-755, (1984). In these reports, human OSE cells that are isolated andcultured are already in terminally differentiated form. The isolationand characterization of OSE cells and granulosa cells in rabbits havealso been reported. Piquette G. N. and Timms B. G. In Vitro Cell.Dev.Biol.26: 471-481, (1990). However, the OSE cells are alreadydifferentiated and therefore, make studying events related to early OSEdevelopment or early cancerous transformation during OSE developmentdifficult. Some research progress has been accomplished towardidentifying precursors to granulosa cells. The number of clonalprecursors of granulosa cells in mouse ovary has been determined to besmall in number (about five) based on a technique of random X-chromosomeinactivation and the use of an X-linked alloenzyme variant ofphosphoglycerate kinase-1 (PGK-1), a variant of a glycolytic enzyme.Telfer E. et. al. J. Reprod. Fert. 84,105-110, (1988). Other researchrelated to ovarian granulosa stem cells demonstrate that granulosacells, not oocytes, are the source of telomerase activity in the ovaryand therefore, as the authors state, their results supports theirhypothesis that granulosa cells arise from a population of stem cells.Lavranos T. C., et. al. Biol. of Reproduction 61, 358-366, (1999). Oneline of thought in ovarian precursor cell research advocates the use offetal, not adult, ovaries as a source of cells because precursor cellsin fetus would presumably have higher activity and be greater in numberthan in adults. To this end, fetal epithelial cells from a region of theovary called rete ovarii have been described but these cells are notdescribed as having pluripotent capacity to differentiate into any othertypes of ovarian mesothelial cells. Dubeau et. al. Anticancer Research10: 1233-1240, (1990).

Accordingly, there exists a need for methods to identify, isolate,culture, and characterize ovarian mesothelial cells that havepluripotent capabilities. The invention described herein overcomes manyof the aforementioned shortcomings and also provides related advantages.

DISCLOSURE OF THE INVENTION

This invention is related to the field of developmental and cellbiology. In one aspect, the invention relates to a population ofsubstantially pure human ovarian mesothelial cells that have thepluripotent capability to differentiate into ovarian surface epithelialcells and granulosa cells.

In another aspect of this invention, the invention relates to methods ofisolating a population of substantially pure human ovarian mesothelialcells that have the pluripotent capability to differentiate into ovariansurface epithelial cells and granulosa cells.

In yet another aspect of this invention, the invention relates tomethods of maintaining a population of substantially pure human ovarianmesothelial cells that have the pluripotent capability to differentiateinto ovarian surface epithelial cells and granulosa cells andmaintaining or culturing these ovarian mesothelial cells such that thecells retain their pluripotent capacity.

In still another aspect of this invention, the invention relates tomethods of providing a source of immunogen,and the uses of asubstantially pure population of ovarian mesothelial cells as animmunogen.

In still another aspect of this invention, the invention relates tomethods of generating a human ovarian tissue model by introducing asubstantially pure population of human ovarian mesothelial cells into anon-human, mammalian recipient.

In another aspect of this invention, the invention relates to methods ofproviding cell therapy whereby a substantially pure population of humanovarian mesothelial cells are introduced into a heterologous recipientin a location which can support the proliferation and growth of theovarian cells.

In another aspect of this invention, the invention relates to methods ofproviding a source of ovarian mesothelial tissue-specific biologicalcomponents for developing pharmaceutical drugs wherein a substantiallypure population of human ovarian mesothelial cells is used as a sourceof ovarian mesothelial biological components in which one or more ofthese ovarian mesothelial biological components are the targets of thedrugs that are being developed.

In another aspect of this invention, the invention relates to methods ofproviding a source of nucleic acids or proteins for bioassay developmentwherein a substantially pure population of human ovarian mesothelialcells is used as a source of nucleic acids or proteins and wherein thesenucleic acids or proteins are used as one or more principal componentsin a bioassay or the development of a bioassay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a microphotograph showing the outgrowth of human ovarianmesothelial cells from solid ovarian tissue. FIG. 1B is amicrophotograph showing the growth of human ovarian mesothelial cells ascell clusters in a suspension culture.

FIG. 1C is a microphotograph showing growth of human ovarian mesothelialcells as a monolayer.

FIG. 2 shows the results of an immunohistochemical analysis whereinmonoclonal antibody 5C8 specifically recognizes ovarian mesothelialcells in human fetal ovarian tissue. FIG. 2A shows magnification at 100×and FIG. 2B shows magnification at 400×.

FIG. 3 shows the results of an immunoperoxidase staining of ovarianmesothelial cell clusters. FIG. 3A shows staining of ovarian mesothelialcells for cytokeratin 19. FIG. 3B shows staining of ovarian mesothelialcells for cytokeratins 13 and 16. FIG. 3C shows staining of ovarianmesothelial cells for cytokeratins 10, 11, and 18. FIG. 3D showsstaining of ovarian mesothelial cells for vimentin. FIG. 3E showsstaining of ovarian mesothelial cells for ovarian mesothelial cellsurface antigen recognized by monoclonal antibody 5C8.

FIG. 4 shows the results of a tissue recombination experiment whereinhuman ovarian mesothelial cells were recombined with rat urogenitalsinus mesenchymal tissue and transplanted into mice. FIGS. 4A, 4B, and4C show the morphology of ovarian surface epithelial cells resemblingcystic structures.

FIG. 5 show the results of immunohistochemical analysis whereincancerous epithelial cell in ovarian cancer can be detected (brown,indicated by arrows) specifically by anti-ovary monoclonal antibody 5C8.

MODES FOR CARRYING OUT THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Thisdetailed description should not be construed to limit the presentinvention, as modifications of the embodiments disclosed herein may bemade by those of ordinary skill in the art without departing from thespirit and scope of the present invention. Throughout this disclosure,various publications, patents, and published patent specifications arereferenced by citation. The disclosure of these publications, patents,and published patents are hereby incorporated by reference in theirentirety into the present disclosure.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA, which are within theskill of the art. See, e.g., Sambrook, et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS INENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J.MacPherson, B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane,eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

Definitions

As used in the specification and claims, the singular form “a”, “an”,and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used in the specification and claims, the terms “ovarian surfaceepithelial cells” and “OSE cells” are interchangeably and refer to“ovarian surface epithelial cells” and “OSE cells” of human origin.

“Ovarian mesothelial cells” refers to human cells derived from themesoderm that have already committed to becoming cells of ovariannature. More specifically, ovarian mesothelial cells of this inventionrefer to cells between the stage of being a mesothelial cell and thestage prior to becoming terminally differentiated to an ovarian surfaceepithelial cell or a granulosa cell. It is at this stage of beingmesothelial cells and just prior to committing to being terminallydifferentiated ovarian mesothelial cell types that the ovarianmesothelial cells of this invention resides. The ovarian mesothelialcells of this invention have a pluripotent or multipotent capacity tobecome either ovarian surface epithelial cells or granulosa cells.

“Pluripotent” and “multipotent” are used interchangeably throughout andrefer to a stage where a cell can still become one of a plurality ofcells but can no longer become any type of cell in the body (i.e. nolonger totipotent). “Pluripotent” cells are not referred to as “stemcells” but rather “progenitor cells” because they are progenitors to oneor more type of a plurality of cells.

As used herein, “pre-determined ovarian” refers to a stage ofdevelopment of a multipotent cell that is prior to the stage of being aprimordial follicle and before the stage of terminally differentiatedovarian cells (such as granulosa cells or ovarian surface epithelialcells). Cells which are “pre-determined ovarian” are committed tobecoming ovarian cells but have not begun to develop into terminallydifferentiated ovarian cells yet. Different factors cause pre-determinedovarian cells to begin differentiating. Non-limiting examples includeexposure to serum, exposure to progesterone, estrogen, luteinizinghormone (LH), contact with surrounding tissue, microenvironment of thecells, and cell-cell contact with surrounding tissue.

An “antibody” is an immunoglobulin molecule capable of binding anantigen. As used herein, the term encompasses not only intactimmunoglobulin molecules, but also anti-idiotypic antibodies, mutants,fragments, fusion proteins, humanized proteins, and modifications of theimmunoglobulin molecule that comprise an antigen recognition site of therequired specificity.

The term “antigen” is a molecule which can include one or more epitopesto which an antibody can bind. An antigen is a substance which can haveimmunogenic properties, i.e., induce an immune response. Antigens areconsidered to be a type of immunogen. As used herein, the term “antigen”is intended to mean full length proteins as well as peptide fragmentsthereof containing or comprising one or a plurality of epitopes.

The terms “surface antigens” and “cell surface antigen” are usedinterchangeably herein and refer to the plasma membrane components of acell. These component include, but are not limited to, integral andperipheral membrane proteins, glycoproteins, polysaccharides, lipids,and glycosylphosphatidylinositol (GPI)-linked proteins. An “integralmembrane protein” is a transmembrane protein that extends across thelipid bilayer of the plasma membrane of a cell. A typical integralmembrane protein consists of at least one membrane spanning segment thatgenerally comprises hydrophobic amino acid residues. Peripheral membraneproteins do not extend into the hydrophobic interior of the lipidbilayer and they are bound to the membrane surface by noncovalentinteraction with other membrane proteins. GPI-linked proteins areproteins which are held on the cell surface by a lipid tail which isinserted into the lipid bilayer.

The term “monoclonal antibody” as used herein refers to an antibodycomposition having a substantially homogeneous antibody population. Itis not intended to be limited as regards to the source of the antibodyor the manner in which it is made (e.g. by hybridoma or recombinantsynthesis). Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen.

“A population of monoclonal antibodies” refers to a plurality ofheterogeneous monoclonal antibodies, i.e., individual monoclonalantibodies comprising the population may recognize antigenicdeterminants distinct from each other.

“Immunogen” refers to any substance that induces an immune response. Asubstance that is an immunogen is described as being “immunogenic”.Induction of immune response includes but is not limited to activationof humoral responses (e.g. producing antibodies) or cellular responses(e.g. priming cytotoxic T cells), inflammatory responses (e.g.recruitment of leukocytes), and secretion of cytokines and lymphokines.

The term “heterologous” as applied to a cell used for immunization ortransplantation means that the cell is derived from a genotypicallydistinct entity from the recipient. For example, a heterologous cell maybe derived from a different species or a different individual from thesame species as the recipient. An embryonic cell derived from anindividual of one species is heterologous to an adult of the samespecies. “Heterologous” as applied to a recipient means that therecipient is a genotypically distinct entity from the source of thecells that are being introduced into the recipient.

“Explant” refers to ovarian tissues taken out of a human fetus.Generally, explants are used as a source of ovarian cells. Isolating thecells from the explant can be accomplished by several methods. Onemethod is to place the ovarian tissue explant, either whole tissue orcut in smaller pieces, in a basal defining media and allow the ovariancells to naturally migrate out of the solid tissue mass into the media.Another method is to subject the ovarian tissue to enzymatic digestionor to mechanical forces that forces cells away from the solid tissue.

A cell is of “ectodermal”, “endodermal” or “mesodermal” origin, if thecell is derived, respectively, from one of the three germlayers—ectoderm, the endoderm, or the mesoderm of an embryo. Theectoderm is the outer layer that produces the cells of the epidermis,and the nervous system. The endoderm is the inner layer that producesthe lining of the digestive tube and its associated organs. The middlelayer, mesoderm, gives rise to several organs, including but not limitedto heart, kidney, mesothelium, and gonads), connective tissues (e.g.,bone, muscles, tendons), and the blood cells.

As used herein, a “substantially pure” population of ovarian mesothelialcells is a population of cells that is comprised at least about 85%ovarian mesothelial cells, preferably at least about 90%, and even morepreferably at least about 95% or more.

The terms “medium”, “cell culture medium”, and “culture medium” are usedinterchangeably. The terms refer to the aqueous microenvironment inwhich the mammalian cells are grown in culture. The medium comprises thephysicochemical, nutritional, and hormonal microenvironment.

A “defined medium,” “basal cell-sustaining medium,” “nutrient medium”,and “basal nutrient medium” are used interchangeably herein and refer toa medium comprising nutritional and hormonal requirements necessary forthe survival and/or growth of the cells in culture such that thecomponents of the medium are known. Traditionally, the defined mediumhas been formulated by the addition of nutritional and growth factorsnecessary for growth and/or survival. Typically, the defined mediumprovides at least one component from one or more of the followingcategories: a) all essential amino acids, and usually the basic set oftwenty amino acids plus cystine; b) an energy source, usually in theform of a carbohydrate such as glucose; c) vitamins and/or other organiccompounds required at low concentrations; d) free fatty acids; and e)trace elements, where trace elements are defined as inorganic compoundsor naturally occurring elements that are typically required at very lowconcentrations, usually in the micromolar range. The defined medium mayalso optionally be supplemented with one or more components from any ofthe following categories: a) one or more mitogenic agents; b) salts andbuffers as, for example, calcium, magnesium, and phosphate; c)nucleosides and bases such as, for example, adenosine and thymidine,hypoxanthine; and d) protein and tissue hydrolysates.

As used herein, “conditioned media” refers to culture media, free ofintact cells, in which ovarian cells have been grown. Ovarian cellsgrown in nutrient media may release factors which promote the continuedsurvival, growth, and maintenance of pre-existing state ofpre-differentiation of the ovarian mesothelial cells. Conditioned mediamay be used to reconstitute a cell pellet or added to cells alreadyexisting in culture plates. Conditioned media may also be used alone orto supplement nutrient media being used to feed ovarian cells.

“Standard incubation conditions” refers to the physicochemicalconditions in an incubator designed for tissue culture in which cellsare placed. Generally, the standard incubation conditions are about 37degrees Celsius and about 5% CO₂ content with humidification. All tissueculture techniques and equipment should be performed under sterileconditions.

Ovarian mesothelial cell clusters”, “ovarian mesothelial cell spheres”,and “ovarian cell clusters” are used interchangeably throughout andrefers to a mass of a plurality of ovarian mesothelial cells. Ovarianmesothelial cell clusters can form a three-dimensional structureresembling roughly a sphere.

A “grafting recombinant”, as used herein, refers to the combined unit ofovarian mesothelial cell clusters placed with mesenchymal tissue.Mesenchymal tissue can be of ovarian or non-ovarian origin. Mesenchymaltissue can be from a species heterologous to the graft recipient.Mesenchymal tissue can also be from a species heterologous to the sourceof ovarian mesothelial cells. Grafting recombinants can be incubated onsubstrate, preferably a soft, biological substrate (e.g. agar) for aperiod ranging from about 1 hour to 96 hours, more preferably betweenabout 6 hours to 48 hours, and even more preferably, overnight with anincubation period of about 24 hours.

“Serum”, as used herein, refers to the fluid phase of mammalian bloodthat remains after blood is allowed to clot.

“Serum biomolecules”, as used herein, refers to biological compositionsfound in serum. Examples include, but are not limited to, albumin,α1-globulin, α2-globulin, β-globulin, and γ-globulin. Serum biomoleculescan include biological compositions, whole or partial, that are eithernaturally found in serum or derived from processing and handling ofserum.

The terms “mammals” or “mammalian” refer to warm blooded vertebrateswhich include but are not limited to humans, mice, rats, rabbits,simians, sport animals, and pets.

Isolation and Maintenance of Human Ovarian Mesothelial Cells

Ovarian mesothelial cells of this invention are isolated from humanfetal ovarian tissue. The age of the fetus is between about week 1 andabout week 40, preferably between about week 8 and about week 30, andeven more preferably between about week 17 and about week 25. Theovarian tissue can be identified by gross anatomy, outward appearance,and location within the fetus. Several features of gross anatomy andappearance distinguishing a ovary are its crescent shape and location inthe abdominal cavity. The ovary may also be associated with fallopiantubes. Once identified, fetal ovarian tissue is first cleaned by washingwith basal nutrient medium and then microdissected. The purposes ofmicrodissection are to remove adjacent tissue to the ovary and to dividethe solid ovarian tissue mass into smaller parts of the whole tissuemass so that the basal nutrient media has greater access to ovariancells within the tissue pieces and/or to separate ovarian cells fromovarian tissue mass. Non-limiting examples of microdissection includedevices that render mechanical shearing forces (i.e. homogenizer, mortarand pestle, blender, etc.), devices that render cuts or tears (i.e.scalpel, syringes, forceps, etc.), or ultrasonic devices. Alternatively,another method of microdissecting fetal ovarian tissue is the use ofenzyme treatment. Various enzyme treatments used to microdissect tissueare well known in the art. One method includes the use ofcollagenase-dispase to digest partially sheared ovarian tissue in abuffered medium that will sustain viability of cells isolated from theovarian tissue. The amount of enzyme will depend on the age of the fetusand how large the ovarian tissue is. In one embodiment, enzyme treatmentwith collagenase-dispase may lower the overall cell yield. Accordingly,the amount of enzyme used would be reduced or not used at all. In otherembodiments, enzyme treatment may increase overall cell yield.Accordingly, enzyme treatment may be used alone or in combination withmicrodissection methods. A wide variety of basal cell-sustaining mediathat can be used to keep the pH of the liquid in a range that promotessurvival of ovarian mesothelial cells and to provide additional volumeof liquid within which the enzymatic digestion can occur. Non-limitingexamples include F12/DMEM, Ham's F10 (Sigma), CMRL-1066, Minimalessential medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's ModifiedEagle's Medium (DMEM, Sigma), and Iscove's Modified Eagle's Medium(IMEM). In addition, any of the basal nutrient media described in Hamand Wallace (1979) Meth. Enz., 58:44, Barnes and Sato (1980) Anal.Biochem., 102:255, or Mather, J. P. and Roberts, P. E. (1998)“Introduction to Cell and Tissue Culture”, Plenum Press, New York canalso be used.

Small pieces of ovarian tissue are placed in a basal cell-sustainingmedia. A variety of basal cell-sustaining media is available for use.Examples include, but are not limited to, Ham's F12 medium, RPMI-1640,and CMRL-1066. For more optimal conditions to promote ovarianmesothelial cell survival and growth, a variety of nutrients may beadded to supplement the basal media. Examples include, but are notlimited to, insulin, transferrin, epidermal growth factor, α-tocopheral,recombinant human heregulin, aprotinin, fetal bovine serum, and bovineserum albumin. In a preferred embodiment, the following amounts ofnutrients are used to promote ovarian mesothelial cell survival andgrowth: at least about 10 ng/ml insulin and not more than about 1 mg/mlinsulin, more preferably about 10 μg/ml insulin; at least about 1 μg/mltransferrin and not more than about 100 μg/ml transferrin, morepreferably about 10 μg/ml transferrin; at least about 1 ng/ml epidermalgrowth factor and not more than about 1000 ng/ml epidermal growthfactor, more preferably about 50 ng/ml epidermal growth factor; at leastabout 0.1 μg/ml α-tocopherol and not more than about 1 mg/mlα-tocopherol, more preferably about 5 μg/ml α-tocopherol; at least about0.1 nM recombinant human heregulin and not more than about 100 nMrecombinant human heregulin, more preferably about 10 nM recombinanthuman heregulin,; at least about 1 μg/ml aprotinin and not more thanabout 100 μg/ml aprotinin, more preferably about 5 μg/ml aprotinin; atleast about 0.1% bovine serum albumin (BSA) and not more than about 50%BSA, more preferably about 2% BSA.

Ovarian mesothelial cells migrate out the ovarian tissue into the mediain which the ovarian tissue is placed. In one embodiment, the ovarianmesothelial cells migrate out of the ovarian tissue into the media incluster form. In another embodiment, the ovarian mesothelial cellsmigrate out of the ovarian tissue into the media in the form of singlecells. In another embodiment, the ovarian mesothelial cells that migrateout of the ovarian tissue are no longer imbedded in the ovarian tissuebut are loosely associated with the tissue. The ovarian mesothelialcells may be grown on different substrates. Non-limiting examples ofsubstrates that may be used include fibronectin, laminin, collagen,polylysine, nitrocellulose, nylon, and polytetrafluoroethylene. In oneembodiment, ovarian mesothelial cells are grown on laminin-coated tissueculture plates in the preferred nutrient media described above. In apreferred embodiment, ovarian mesothelial cells are grown inlaminin-coated tissue flasks in the preferred nutrient media describedabove. The size of the tissue culture plates or flasks are proportionalto the amount of ovarian tissue being placed within the plates orflasks. A skilled artisan may determine the correct size of the flask orplate by a stepwise increment of ovarian tissue placed within the tissueculture plates or flasks. When the ovarian tissue is first placed withinthe plates or flasks, the media is generally clear in overall turbidity.As ovarian mesothelial cells migrate out and away from the ovary tissuepieces, the media will become more opaque and more turbid. At the pointwhere the media is highly turbid because of the increasing amount ofovarian mesothelial cells migrating from the ovarian tissue or becauseof ovarian mesothelial cell growth, more nutrient media is placed in theplates or flasks to replenish the nutrients consumed by the ovariancells. In the alternative, when the media becomes turbid with increasingamounts of ovarian mesothelial cells, a small amount of cells may beremoved from the culture plates or flasks and checked for cellviability, for example, with trypan blue staining. Plates or flasks thathave been overrun with too many cells will begin to show decreased cellviability. The skilled artisan may then transfer the contents of theplates or flasks to other plate or flasks of a larger size (e.g. greatercubic volume) to accommodate the increasing amount of cells. In oneembodiment, the entire content of the plate or flask is transferred toanother plate or flask of a larger cubic volume. In another embodiment,the ovarian cell suspension is split into several parts, each part isplaced in a separate plate or flask and then nutrient media is added tothe ovarian cells (also known as “subculturing”). Ovarian mesothelialcells form cell clusters when cultured in the preferred nutrient mediain laminin-coated flasks. The culturing combination of tissue cultureflasks and laminin coating allows for separation of mesenchymal cellsand ovarian mesothelial cell clusters. Mesenchymal cells migratethroughout the volume of the media while ovarian mesothelial cellclusters associate in close physical proximity with the ovarian tissue.In another embodiment, ovarian mesothelial cells are grown in thepreferred nutrient media described above in laminin-coated tissueculture plates. Ovarian mesothelial cells form monolayers in thisembodiment.

Ovarian mesothelial cells cultured in the preferred nutrient media inlaminin-coated tissue culture flasks will form cell clusters that are inclose physical proximity with ovarian tissue. When enrichment of theovarian mesothelial cells is desired, one method that may be employed isenzymatic treatment to dissociate ovarian mesothelial cells followed byisolation of the ovarian mesothelial cell clusters. Examples of enzymesthat can be used in enzymatic treatment include but are not limited tocollagenase-dispase and trypsin. In one embodiment, collagenase-dispaseis used to dissociate ovarian mesothelial cell clusters from cultureflask walls and from ovary tissue, preferably at least about 10% (w/v)is used, more preferably at least about 1% of collagenase-dispase isused, most preferably at least about 0.1% of collagenase-dispase isused. Ovarian mesothelial cell clusters are isolated by using a densitygradient. Compounds that can be used to achieve cell separation include,but are not limited to, serum (i.e. bovine serum albumin or BSA),ovalbumin, nonionic synthetic polymers of sucrose (i.e. Ficoll™),colloidal polyvinylpyrrolidone-coated silica (i.e. Percoll™),polyvinylpyrrolidone or PVP, and methylcellulose. In a preferredembodiment, density gradients that are capable of neutralizingcollagenase-dispase are used. One example of such a density gradient isBSA. The amount of BSA used is about 50% volume-to-volume ratio with thepreferred nutrient media, more preferably about 25%, more preferablyabout 10%, more preferably at least about 0.1%, and most preferablyabout 1% to 3%. In some cases, one density gradient is sufficient toenrich a population of ovarian mesothelial cells. In other cases, morethan one application of a density gradient will be needed. The desiredproduct is a population of substantially pure ovarian mesothelial cellclusters.

In one embodiment, a substantially pure population of ovarianmesothelial cell clusters is isolated by spinning ovarian cells througha density gradient like BSA at a spinning rate sufficient to pellet theovarian mesothelial cell clusters and leave single mesenchymal cells inthe supernatant. The cell pellet with ovarian mesothelial cells isresuspended in a nutrient media sufficient to sustain life of theovarian mesothelial cells and placed in a tissue culture flask which iscoated with a biological substrate, for example laminin. The entirety ofthe volume of resuspended ovarian mesothelial cells may be placed in oneflask. In an alternative, if the resuspended ovarian mesothelial cellsis highly enriched and has a high density of ovarian mesothelial cells,then the volume of resuspended ovarian mesothelial cells may be splitinto several different flasks to which the preferred nutrient media isadded. In a preferred embodiment, the following amounts of nutrients areused to promote the survival and growth of the substantially purepopulation of ovarian mesothelial cells: at least about 10 ng/ml insulinand not more than about 1 mg/ml insulin, more preferably about 10 μg/mlinsulin; at least about 1 μg/ml transferrin and not more than about 100μg/ml transferrin, more preferably about 10 μg/ml transferrin; at leastabout 1 ng/ml epidermal growth factor and not more than about 1000 ng/mlepidermal growth factor, more preferably about 50 ng/ml epidermal growthfactor; at least about 0.1 μg/ml α-tocopherol and not more than about 1mg/ml α-tocopherol, more preferably about 5 μg/ml α-tocopherol; at leastabout 0.1 nM recombinant human heregulin and not more than about 100 nMrecombinant human heregulin, more preferably about 10 nM recombinanthuman heregulin; at least about 1 μg/ml aprotinin and not more thanabout 100 μg/ml aprotinin, more preferably about 5 μg/ml aprotinin; atleast about 0.1% bovine serum albumin (BSA) by volume and not more thanabout 50% BSA, more preferably about 0.5% BSA.

The frequency of feeding ovarian mesothelial cells may be once a day orevery other day. In one embodiment, ovarian mesothelial cells may be fedby replacing the entirety of the old nutrient media with new nutrientmedia. In another embodiment, ovarian mesothelial cells may be fed withconditioned media in which these cells were grown. Because the claimedovarian mesothelial cells are unique to this invention and will secretefactors specific to these cells, the conditioned media derived from theovarian mesothelial cells are also unique. In this invention, ovarianmesothelial cells form clusters when grown in the preferred nutrientmedia, defined above, in laminin-coated tissue culture flasks. When thesubstrate is laminin-coated tissue culture plates, ovarian mesothelialcells form an attached stromal monolayer. In a preferred embodiment ofthe invention, cell to cell contact of ovarian mesothelial cells to eachother is maintained throughout the culturing of ovarian mesothelialcells to promote a higher proliferation rate. Addition of conditionedmedia may also promote better growth of the ovarian mesothelial cells. Askilled artisan can determine if the addition of conditioned media isadvantageous to the growth of ovarian mesothelial cells by supplementingthe nutrient media stepwise with an increasing amount of conditionedmedia. Cell growth can be determined by counting the number of cells pervolume of media before and after the addition of conditioned media.Alternatively, cell viability (e.g. trypan blue) can be used to assessif addition of conditioned media to the culturing condition isadvantageous to the growth of the ovarian mesothelial cells. A frequencyof feeding that is preferable for promoting the survival and growth ofovarian mesothelial cells is once a week, even more preferably is twicea week, and most preferably every other day. The ovarian mesothelialcells of this invention can be passaged multiple times without inducingdifferentiation of these ovarian mesothelial cells into terminallydifferentiated ovarian surface epithelial cells and granulosa cells.

Characterization of Ovarian Mesothelial Cells

The population of ovarian mesothelial cells of this invention isolatedin the manner disclosed herein have several defining characteristics.First, the ovarian mesothelial cells are at a stage that can bedescribed as “pre-determined ovarian”. Of the mesothelial progenitorcells, some are predetermined to become ovarian mesothelial cells. It isat this stage of development that the population of ovarian mesothelialcells claimed herein resides. The ovarian mesothelial cells of thisinvention have the capacity to become either ovarian surface epithelialcells and granulosa cells but have not begun to differentiate intoeither cell type.

Identification of ovarian mesothelial cells may be accomplished bymorphology or specific markers or a combination of both techniques.Granulosa cells line form the wall of an ovarian follicle. Morphology ofgranulosa cells is a cuboidal shape. Markers that can be used to detectovarian mesothelial cells include but are not limited to cytokeratin(CK) 1, 5, 6, 7, 8, 10, 11, 13, 15, 16, 18, and 19, anti-ovarymonoclonal antibody 5C8, and vimentin on ovarian mesothelial cellsurfaces. Examples of antibodies specific for CK and vimentin that maybe used include, but are not limited to: anti-cytokeratin (CK)antibodies clone 4.62, clone 8.12, clone 8.13 from Sigma Chemical Co.and anti-vimentin antibodies clone 13.2 from Sigma Chemical Co. Anti-CKantibodies and anti-vimentin antibodies can be used in either direct orindirect staining of MTE cells in immunohistochemistry or by flowcytometry. Markers to detect ovarian mesothelial cells can be used indirect and indirect immunofluorescence, immunohistochemistry, and flowcytometry.

Ovarian mesothelial cells of this invention are maintained at theirpreexisting pre-differentiation state in basal nutrient media. Basalcell-sustaining media or the preferred nutrient media disclosed hereinor conditioned media may be used to culture the ovarian mesothelialcells in vitro. Different types of substrate on tissue culture platescan be used to obtain either clusters or monolayers of ovarianmesothelial cells. The use of laminin-coated flasks in conjunction withthe preferred nutrient media disclosed herein results in clusters ofovarian mesothelial cells whereas the use of laminin-coated plates inconjunction with the preferred nutrient media disclosed herein resultsin monolayers of ovarian mesothelial cells. Ovarian mesothelial cells ofthis invention may be cultured in serum-free nutrient media orserum-containing nutrient media. As is well-known to those of ordinaryskill in the art, serum is commonly added to nutrient media to furtherenhance cell growth. Serum contains many serum biomolecules, however,the ovarian mesothelial cells of this invention may be grown in theabsence of a plurality of these serum biomolecules. Cell growth ofovarian mesothelial cells may be enhanced by the addition of one or moreproteins found in serum, for example, bovine serum albumin (or BSA).

Ovarian mesothelial cells of this invention have the capacity to bepassaged multiple times in the preferred basal nutrient media disclosedherein. Multipotency is retained during each passage and at any pointafter each passage, ovarian mesothelial cells of this invention candifferentiate into ovarian surface epithelial cells and granulosa cells.In addition, at any point after each passage, ovarian mesothelial cellsmay be used as an immunogen, for cell therapy, for bioassays, toestablish a human ovarian model, or for drug discovery and/ordevelopment as disclosed herein.

Another characteristic of the ovarian mesothelial cells of thisinvention is the capacity to differentiate into ovarian surfaceepithelial cells and granulosa cells upon transplantation under kidneycapsule of a recipient mammal. Prior to transplantation, ovarianmesothelial cells have the capacity to differentiate into granulosacells or ovary surface epithelial cells. As disclosed herein, ovarianmesothelial cells can be grown either in ovarian mesothelial cellclusters or in monolayers and then combined with mesenchymal tissue andplaced under a kidney capsule of a recipient mammal. Preferably, humanovarian mesothelial cell clusters are combined with rat urogenitalmesenchymal tissue and placed under the kidney capsule of a recipientmammal. A portion of the transplant may be removed for analysis usingthe markers, morphology, or a combination thereof to identify theovarian cells.

Uses of Ovarian Mesothelial Cells

Uses as an Immunogen

One use for ovarian mesothelial cells is as an immunogen. Ovarianmesothelial cells isolated and cultured with the methods disclosedherein can be used as an immunogen that is administered to aheterologous recipient. Administration of ovarian mesothelial cells asan immunogen can be accomplished by several methods. Methods ofadministrating ovarian mesothelial cells as immunogens to a heterologousrecipient include but are not limited to: immunization, administrationto a membrane by direct contact such as swabbing or scratch apparatus,administration to mucous membrane by aerosol, and oral administration.As is well-known in the art, immunization can be either passive oractive immunization. Methods of immunization can occur via differentroutes which include but are not limited to intraperitoneal injection,intradermal injection, local injection. Subjects of immunization mayinclude mammals such as mice. The route and schedule of immunization aregenerally in keeping with established and conventional techniques forantibody stimulation and production, for example, injecting immunogen infoot pads of mice once a week for several weeks. While mice are employedin this embodiment, any mammalian subject including humans or antibodyproducing cells therefrom can be manipulated according to the processesof this invention to serve as the basis for production of mammalianhybridoma cell lines. Typically, mice are inoculated with an immunogenicamount of the ovarian mesothelial cells and then boosted with similaramounts of the immunogen. In an alternative, cells grown onnon-biological membrane matrix, are surgically implantedintraperitoneally into the host mammal. Lymphoid cells, preferablyspleen lymphoid cells from the mice, are collected a few days after thefinal boost and a cell suspension is prepared therefrom for use in thefusion.

Hybridomas are prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 as modified by Buck, D.W., et al., (1982) In Vitro, 18:377-381. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. The technique involves fusing the myeloma cellsand lymphoid cells using a fusogen such as polyethylene glycol, or byelectrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as HAT medium, to eliminate unhybridizedparent cells. Any of the media described herein can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells areused to produce the monoclonal antibodies of the subject invention. Thehybridomas are expanded and subcloned, if desired, and supernatants areassayed for anti-immunogen activity by conventional immunoassayprocedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescenceimmunoassay).

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen.

In this manner, a panel of novel antibodies to cell surface antigenspecific to a stage of ovarian mesothelial cells can be generated usingthe ovarian mesothelial cells of this invention as an immunogen. Oncemonoclonal antibodies to cell surface antigens on ovarian mesothelialcells are made by the method disclosed herein, the antibodies can beused for several uses. The antibodies may be sequenced and cloned forpurposes of generating recombinant antibodies or humanized antibodies.Other uses of ovarian mesothelial cell-specific antibodies include, butare not limited to, biological testing and purification (i.e. isolationof ovarian mesothelial cells, for example by flow cytometry or antibodypanning), therapeutic uses (i.e. promoting or arresting cell growth bybinding of antibody to target cell or promoting or arresting growth of acell mass by binding of antibody to target cell), biological markers(i.e. identification of other ovarian or non-ovarian cells), clinicaldiagnosis (i.e. identification of cancerous ovarian cells in tissuesamples).

Another use as an immunogen is to modulate overall immune response in aheterologous recipient. As is well-documented in the art, foreignsubstances such as cells or organs introduced into a heterologousrecipient may induce a variety of immune responses. The immune responsescan be in the form of rejection (e.g. in organ transplantation), T cellactivation (e.g. cross-priming), anergy, or tolerance. The overallimmune response can be systemic or localized. In the case where alocalized immune response is desired, for example in the gonadal region,an immunogen such as ovarian mesothelial cells is introduced into thegonadal region in an effective amount. Effective amount can bedetermined in a stepwise fashion in which increasing amounts of ovarianmesothelial cells are introduced into a heterologous recipient and thesubsequent immune response is monitored. Overall immune response (e.g.antibody production, cytokine production, T cell proliferation, anergy,tolerance, etc.) may be monitored by a number of methods including butnot limited to ELISA, proliferation assays, flow cytometry with cellsurface markers, and immunohistochemistry.

Use of Ovarian Mesothelial Cells for Drug Discovery

Another use of ovarian mesothelial cells is related to drug discovery.Since the multipotent pre-determined ovarian mesothelial cell populationhas not been isolated and cultured in the disclosed manner, the ovarianmesothelial cell population may secrete proteins that have not beenheretofore discovered or characterized. Therefore, proteins secreted byovarian mesothelial cells may be used as targets for drug development.In one embodiment, drugs can be made to target specific proteins onovarian mesothelial cells in vivo. Binding of the drug may promotedifferentiation of the ovarian mesothelial cells into ovarian surfaceepithelial cells or granulosa cells. This approach may be useful whenovarian surface epithelial cell or granulosa cell neogenesis is desired,for example to replace damaged cells after cancer therapy (e.g.chemotherapy, radiation therapy, etc.). In another embodiment, drugspecific for regulatory proteins of ovarian mesothelial cells may beused to arrest growth of a particular type of cell, for example in casesof cancer (i.e. ovarian cancer, uterine cancer, etc.).

Uses of Ovarian Mesothelial Cells for Cell Therapy

In another use, ovarian mesothelial cell lines are used for celltherapy. Transplantation of ovarian mesothelial cells is one suchexample of cell therapy. In cases where different types of ovarian cells(i.e. OSE cells or granulosa cells) are desired, transplantation ofovarian mesothelial cells may be employed because the ovarianmesothelial cells of this invention are multipotent and candifferentiate into ovarian surface epithelial cells and granulosa cells.To practice this use, ovarian mesothelial cells are isolated andcultured in basal nutrient, nutrient-defined media using the methodsdisclosed. Ovarian mesothelial cells are grown on laminin-coated tissueculture flasks to obtain ovarian mesothelial cell clusters. Ovarianmesothelial cell clusters are grown under standard incubation conditionsfor about half a day to at least about 1 cell cycle passage, morepreferably for at least about 2 cell cycle passage, most preferably atleast about 3 cell cycle passages. Ovarian mesothelial cell aggregatescan then be administered to a recipient and allowed to differentiate. Inan alternative, ovarian mesothelial cell aggregates can be used ascellular carriers of gene therapy wherein ovarian mesothelial cells aretransfected with one or more genes and enclosed in a delivery device andthen administered to a recipient. In another embodiment, ovarianmesothelial cell aggregates are placed under a kidney capsule andallowed to differentiate into OSE or granulosa cells. In anotherembodiment, ovarian mesothelial cell aggregates are used in a devicewhich contains cells and limits access from other cells (i.e.Theracyte®) to limit immune system responses.

Uses of Ovarian Mesothelial Cells to Make Human Tissue Models

Another use for ovarian mesothelial cells is to generate human ovarytissue models in non-human mammals. A human ovary tissue models can beemployed to study multiple facets of ovary development or ovarycarcinogenesis, an important area of ovarian cancer research. Ovarianmesothelial cell spheres are placed on top of mesenchymal tissue to formgrafting recombinants. To form grafting recombinants, about 1 to 15ovarian mesothelial cell spheres, more preferably about 5 to 8 spheres,are placed on top of mesenchymal tissue. The mesenchymal tissue may beeither ovarian or non-ovarian tissue and may be derived from a differentspecies from which ovarian mesothelial cells are isolated. In a workingexample, human ovarian mesothelial cells are placed on top of ratmesenchymal urogenital tissue to form a graft recombinant. A skilledartisan may determine the optimal combination for human ovarianmesothelial cell growth in a stepwise fashion, by first isolating humanovarian mesothelial cells using the methods disclosed herein and thencombining with mesenchymal tissue from different organs. In someembodiments, a different species, e.g. rat, is used as a source formesenchymal tissue in combination with human ovarian mesothelial cells.The use of heterologous species allows human-specific markers to be usedto determine the identity of differentiated human ovarian cells. Thelikelihood of false positives is reduced if rat mesenchymal tissue isused. Likewise, the use of urogenital mesenchymal tissue over ovarianmesenchymal tissue reduces the likelihood of false positives inidentifying differentiated ovarian cells. In a preferred embodiment,about 1 to 12 ovarian mesothelial cell spheres, even more preferablyabout 5 to 8 ovarian mesothelial cell spheres, are placed on top of raturogenital mesenchymal cells. Preferably, about 1×10⁴ to about 5×10⁶mesenchymal cells are used. Even more preferably, about 2×10⁵ to about5×10⁵ mesenchymal cells are used. A graft recombinant comprising ovarianmesothelial cell spheres placed on mesenchymal tissue is then placedunder the kidney capsule of a recipient mammal. Possible recipientmammals include but are not limited to mice and rats. Typically in graftsituations, donor tissue is vulnerable to attack by the recipient'simmune system. To alleviate graft rejection, several techniques may beused. One method is to irradiate the recipient with a sub-lethal dose ofradiation to destroy immune cells that may attack the graft. Anothermethod is to give the recipient cyclosporin or other T cellimmunosuppressive drugs. With the use of mice as recipient mammals, awider variety of methods are possible for alleviating graft rejection.One such method is the use of an immunodeficient mouse (nude or severecombined immunodeficiency or SCID). In a working example, human ovarianmesothelial cell spheres are placed on rat urogenital mesenchymal tissueand placed under the kidney capsule of an immunodeficient mouse. Thegraft recombinant remains in the recipient for about 1 to about 52weeks, preferably about 5 to about 40 weeks, and even more preferablyabout 6 to about 8 weeks before the grafts are harvested and analyzedfor ovarian mesothelial cell differentiation. In some cases, a smallportion of the graft is needed for analysis. Markers specific for theovarian surface epithelial cell and granulosa cells include, but are notlimited to, cytokeratins 1, 5, 6, 7, 8, 10, 11, 13, 15, 16, 18, and 19,follicle stimulating hormone (FSH) receptor, luteinizing hormone (LH)receptor, and aromatase) may be utilized to confirm the identity of thedifferentiated ovarian mesothelial cells. Non-limiting methods ofconfirming markers are immunohistochemical analysis, immunofluorescence,and flow cytometry. Another method of identifying the differentiatedovarian mesothelial cells and assessing the success of thetransplantation is to stain for the presence of 17β-hydroxysteroiddehydrogenase (17β-HSDH) in ovarian surface epithelial cells (OSE) ordelta 5-3β-hydroxysteroid dehydrogenase presence in granulosa cells.These markers can be used separately or in combination with each other.In addition, a combination of one or more of these markers may be usedin combination with cell morphology to determine the efficacy of thetransplantation.

In one embodiment, human ovarian model can be generated in a SCID(severe combined immunodeficiency) mouse. The human ovarian model can bemade by utilizing the human ovarian mesothelial cells isolated andcultured with methods disclosed herein and using the human ovarianmesothelial cells to make graft recombinants. Graft recombinants arethen placed under the kidney capsule of mice. After about 1 to 10 weeks,preferably about 6 to 8 weeks after implantation under the kidneycapsule, the graft or portion thereof is harvested and analyzed byimmunohistochemistry. Markers specific to ovarian surface epithelialcells and granulosa cells include, but are not limited to, cytokeratin(i.e. CK 1, 5, 6, 7, 8, 10, 11, 13, 15, 16, 18, and 19), folliclestimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor,and aromatase. Markers specific to ovarian surface epithelial cells andgranulosa cells are used to analyze the efficacy of the tissue modelsystem. Alternatively, markers specific for differentiated ovarianmesothelial cells are used. Non-limiting examples of these markers are:cytokeratin-19, vimentin, and monoclonal antibody 5C8. Yet another wayto assess the results of ovarian mesothelial cell differentiation is bymorphology. Ovarian surface epithelial cells have the appearance of flator columnar epithelial cells.

Uses of Ovarian Mesothelial Cells in Bioassays

The ovarian mesothelial cells disclosed herein can be used in variousbioassays. In one use, the ovarian mesothelial cells are used todetermine which biological factors are required for differentiation. Byusing the ovarian mesothelial cells in a stepwise fashion in combinationwith different biological compounds (such as hormones, specific growthfactors, etc.), one or more specific biological compounds can be foundto induce differentiation of ovarian mesothelial cells to OSE cells.Employing the same stepwise combinations, one or more specificbiological compound can be found to induce differentiation of ovarianmesothelial cells to granulosa cells. Other uses in a bioassay forovarian mesothelial cells are differential display (i.e. mRNAdifferential display) and protein-protein interactions using secretedproteins from ovarian mesothelial cells. Protein-protein interactionscan be determined with techniques such as yeast two-hybrid system.Proteins from ovarian mesothelial cells can be used to identify otherunknown proteins or other cell types that interact with ovarianmesothelial cells. These unknown proteins may be one or more of thefollowing: growth factors, hormones, enzymes, transcription factors,translational factors, and tumor suppressors. Bioassays involvingovarian mesothelial cells and the protein-protein interaction thesecells form and the effects of protein-protein or even cell-cell contactmay be used to determine how surrounding tissue, such as mesenchymaltissue, contributes to ovarian mesothelial cell differentiation.

EXAMPLES Example 1 Isolation and Culturing of Ovarian Mesothelial Cells

Human fetal ovaries of gestational age between 17 to 25 weeks wereobtained from Advanced Bioscience Research at Alameda county, Calif.Ovaries were procured and shipped to the lab in tissue culture mediumunder wet ice bath. Immediately upon arrival, the ovaries were cleanedof excess connective tissues, carefully separated from fallopian tube,and washed five times with fresh tissue culture medium.

The ovaries were minced with scissors or cut into small pieces (lessthan 1 mm thick) with a razor blade. The tissue pieces from each ovarywere plated directly in a T75 flask freshly coated with laminin with 10ml preferred nutrient medium as disclosed herein. Further dissociationof the ovaries with collagenase-dispase (0.5%) for 30 minutes at 37° C.could be done, but the procedure reduced the recovery of ovarianmesothelial cells. The cells were cultured in F12/DMEM supplemented with10 μg/ml insulin, 10 μg/ml transferrin, 5 μg/ml α-tocopherol, 10 nMrecombinant human heregulin β1, 50 ng/ml epidermal growth factor, 5μg/ml aprotinin, and 2% BSA (by volume) at standard incubationconditions. Within a week, cells migrated from the explant andproliferated to form confluent cell cultures. The cultures at this stagewere consisted both ovarian mesothelial cells and mesenchymal cells.Mesenchymal cells migrated all over the flask, but ovarian mesothelialcells remained in close proximity to the tissue explants (FIG. 1A). Toenrich the ovarian mesothelial cells, the cultures were detached fromthe culture flask by incubation with 0.1% collagenase-dispase for 30minutes. The treatment dissociated the mesenchymal cells into singlecells but left the ovarian mesothelial cells in the clusters. Theovarian mesothelial cells were then separated by centrifugation throughBSA gradients (1%-3% BSA in F12/DMEM) for 5 minutes. The supernatantwhich contained mostly mesenchymal cells was aspirated and the cellpellet remaining contained mostly ovarian mesothelial cells. The ovarianmesothelial cells were resuspended in the culture medium and plated at adilution factor of 1:5 (1 part cell suspension: 5 part total suspensionvolume). The culture medium was F12/DMEM supplemented with 10 μg/mlinsulin, 10 μg/ml transferrin, 5 μg/ml α-tocopherol, 10 nM recombinanthuman heregulin β1, 50 ng/ml epidermal growth factor, 5 μg/ml aprotinin,and 0.5% BSA (by volume). The cells grew in suspension as clusters (FIG.1B). The cells were propagated this way for about 4 to 5 passages. Thecells grown in suspension culture were able to attach to the sides ofthe plate and grew as ovarian mesothelial cell monolayer (FIG. 1C) whenserum was present in the culture medium or when the plates were coatedwith laminin. Monolayer cultures passage well when the ovarianmesothelial cells are kept in contact with each other.

Example 2 Characterization of Ovarian Mesothelial Cell Lines

Ovarian mesothelial cells were harvested from suspension cultures. Todirectly stain the cells in cluster form, the cell clusters wereembedded in optimal cutting temperature (OCT) compound and frozen on dryice. Cryosections (5-10 μm thick) were cut of the OCT blocks,thaw-mounted onto coverslips, and fixed in 3% paraformaldehyde for 1hour. A monolayer culture of ovarian mesothelial cells was prepared byplating the cell clusters from the suspension culture on chamber slidesin the presence of 2% BSA. The monolayer cell cultures were fixed insitu with 3% paraformaldehyde for 1 hour. After the fixative was washedaway with phosphate buffered saline (PBS), the cells were incubated inblocking buffer (5% goat serum and 0.1% Tween 20 in PBS) for 30 minutes,rinsed with PBS, then incubated in primary antibodies for 1 hour, rinsedwith PBS, and then incubated with anti-mouse Ig-horseradish peroxidasefor 1 hour. The markers and exemplary antibodies used to characterizeovarian mesothelial cells were CK 19 and anti-cytokeratin antibody clone4.62 from Sigma Chemical Company (FIG. 3A), CK 13 and CK 16 and antibodyclone 8.12 from Sigma Chemical Company (FIG. 3B), CK 10, CK 11, and CK18 and antibody clone 8.13 from Sigma Chemical Company (FIG. 3C),vimentin and anti-vimentin monoclonal antibody clone 13.2 from SigmaChemical Company (FIG. 3D), and anti-ovarian epithelial monoclonalantibody 5C8 (FIG. 3E). Monoclonal antibody 5C8 was made by usingovarian mesothelial cells as an immunogen.

Example 3 Method of Providing a Source of an Immunogen to ProduceMonoclonal Antibodies Against Ovarian Mesothelial Cells

Ovarian mesothelial cells were isolated and cultured as disclosed andthen subsequently used as an immunogen to produce a panel of monoclonalantibodies against ovarian mesothelial cells. Mice were each immunizedwith one injection of about 1×10⁶ cells of ovarian mesothelial cells perweek. The immunization was localized into the foot pads of each mice.After 5 injections, the mice were bled to check the titer of ovarianmesothelial cells. When the titer of ovarian mesothelial cells roseabove 1:1000 (ovarian cell:total cells) in a mouse, the mouse wassacrificed and lymph nodes were harvested. Lymphocytes were preparedfrom the lymph nodes and fused to myeloma cell line to produce ahybridoma. Hybridomas were screened by flow cytometry(fluorescence-activated cell sorter or FACS) as well as byimmunohistochemistry for monoclonal antibodies that specifically boundto ovarian mesothelial cells. Clonal hybridoma cell lines were obtainedby repeated limiting dilutions. Hybridoma clone 5C8 was a monoclonalantibody clone that bound to ovarian mesothelial cells. FIG. 2 show theresults in an immunohistochemical analysis where monoclonal antibodyclone 5C8 specifically recognized ovarian mesothelial cells in fetalhuman ovaries.

Example 4 Use of Ovarian Mesothelial Cells to Generate a Human OvarianTissue Model

Tissue graft recombinants were made for the purposes of transplantationinto a heterologous recipient either to generate a human ovarian tissuemodel in a heterologous recipient. To make tissue graft recombinants,ovarian mesothelial cells harvested from monolayer cultures after 3passages was placed on top of rat urogenital sinus mesenchymal tissue.The graft recombinant was cultured on an agar plate for about 24 hours.Then the graft recombinant was implanted under the kidney capsule innude mice. The implant was allowed to grow for about 2 months before thegraft recombinant tissues were excised and analyzed by histology. Theresults showed that the ovarian mesothelial cells formed cysticstructures (FIGS. 4A, 4B, and 4C). The epithelial cell lining of theovarian surface epithelial cells was a thin sparse layer, looselyattached cells in some of the cysts (FIGS. 4A, 4B, and 4C).

Example 5 Use of Ovarian Surface Epithelial Cells in Bioassays

By using the ovarian mesothelial cells of this invention as immunogens,monoclonal antibodies specific to ovarian mesothelial cells such asclone 5C8 were generated. These antibodies were used to stain frozensections of ovarian tissue from a female patient with ovarian cancer.Monoclonal antibody 5C8 showed strong staining of cancerous epithelialcells in a patient with ovarian cancer (FIG. 5). Thus the utility ofmonoclonal antibodies such as clone 5C8 can be extended to diagnosticpurposes in a clinical setting.

1. A method of isolating a substantially pure population of ovarianmesothelial cells, comprising: (a) microdissecting a source of humanfetal ovarian mesothelial cells; (b) placing the source of ovarianmesothelial cells in nutrient media under culture conditions sufficientto sustain life of said ovarian mesothelial cells and wherein thenutrient media contains nutrients consisting of insulin, transferrin,epidermal growth factor, α-tocopherol, recombinant human heregulin β1,bovine serum albumin, and aprotinin; (c) maintaining suitable cultureconditions sufficient to allow the migration of ovarian mesothelialcells from the source of ovarian mesothelial cells into the nutrientmedia; (d) maintaining suitable culture conditions sufficient to allowovarian mesothelial cells to form aggregate or monolayer formations; and(e) subculturing said aggregate or monolayer formations to obtain asubstantially pure population of ovarian mesothelial cells.
 2. A methodof isolating a substantially pure population of ovarian mesothelialcells which have a pluripotent capacity to differentiate into ovarysurface epithelial cells or granulosa cells, comprising the steps of:(a) microdissecting a source of human fetal ovarian mesothelial cells;(b) placing the source of ovarian mesothelial cells in nutrient mediaunder culture conditions sufficient to sustain life of said ovarianmesothelial cells and wherein the nutrient media contains nutrientsconsisting of insulin, transferrin, epidermal growth factor,α-tocopherol, recombinant human heregulin β1, bovine serum albumin, andaprotinin; (c) maintaining suitable culture conditions sufficient toallow the migration of ovarian mesothelial cells from the source ofovarian mesothelial cells into the nutrient media; (d) maintainingsuitable culture conditions sufficient to allow ovarian mesothelialcells to form aggregate or monolayer formations; and (e) subculturingsaid aggregate or monolayer formations to obtain an isolated,substantially pure population of ovarian mesothelial cells which have apluripotent capacity to differentiate into ovary surface epithelialcells or granulosa cells.